Sequence information on the human genome and that of selected organisms is becoming available at an ever increasing rate and is providing unparalleled understanding of the complexity of the proteins in cells. The next challenge is at the level of proteomics, understanding the structure and function of proteins encoded by a particular genome. This information will certainly provide the starting point for development of novel therapeutic interventions against many of the world's diseases. Proposed here is research to develop fully automated mass spectrometric methods for the rapid characterization of known and unknown proteins at the low femtomole or attomole level in complex mixtures. Multistage chromatography in conjunction with automated peak parking technology plus nanoliter/min, high performance liquid chromatography and high performance capillary electrophoresis interfaced to ion trap and Fourier tranform mass spectrometers via an electrospray ionization source will be employed in this effort. Specific aims are to characterize: (a) the proteins that transport unspliced mRNA out of the nucleus; (b) the proteins that facilitate splicing of pre-mRNA; (c) proteins required for mitotic chromosome condensation in yeast; (d) a virulence transcription factor in the enteric protozoan, Entamoeba histolytica; (e) the proteins uniquely synthesized in neuronal dendrites and thus assumed to be involved in intracellular signaling, neural plasticity and long term potentiation (memory); (f) the L-selectin glycoprotein ligand that facilitates migration of leucocytes to sites of inflamation; (g) proteins found uniquely on the surface of human sperm that can be used as targets for the development of human contraceptive vaccines; (h) platelet phosphoproteins synthesized in response to thrombin; (i) the post translational modifications on the androgen receptor and the associated proteins that regulate its activity; the proteins that regulate development of the endoderm (internal organs) in the nematode, C. elegans.